信号传导与转录活化因子3抑制剂 WP1066影响人舌鳞状细胞癌增殖与凋亡的研究  

Signal transducer and activator of transcription-3 inhibitor WP1066 affects human tongue squamous cell carcinoma proliferation and apoptosis in vitro and in vivo

作　　者：黄媛媛[1] 周旋[1] 刘爱芹[1] 李莎莎[1] 王旭东[1] 张仑[1] 

机构地区：[1]天津医科大学肿瘤医院颌面耳鼻喉肿瘤科,国家肿瘤临床医学研究中心 天津市肿瘤防治重点实验室,300060

出　　处：《中华口腔医学杂志》2014年第5期308-313,共6页Chinese Journal of Stomatology

基　　金：国家自然科学基金（81101916、81172573）;天津市应用基础及前沿技术研究计划（11JCYBJC10800）

摘　　要：目的探讨信号传导与转录活化因子3（signal transducer and activator of transcription 3， STAT-3）的小分子抑制剂WP1066影响人舌鳞状细胞癌细胞系Tscca增殖与凋亡的分子机制。方法实验共分3组：空白对照组、二甲基亚砜组（ DMSO组）、小分子抑制剂组（ WP1066组）。采用WP1066拮抗磷酸化STAT-3（ p-STAT-3）表达；实时定量PCR 检测加入DMSO、WP1066后微RNA-21表达；甲基噻唑基四唑（ methyl thiazolyl tatrozolium ，MTT）法检测细胞生存率；流式细胞术测细胞早期凋亡；蛋白质印迹法检测 STAT-3及 p-STAT-3、细胞程序死亡蛋白4（ programmed cell death protein 4， PDCD-4）、细胞增殖核抗原（antigen 67，Ki-67）、B细胞淋巴瘤2（B cell lymphoma 2，Bcl-2）、活化型含半胱氨酸的天冬氨酸蛋白水解酶3（cleaved cysteinyl aspartate specific proteinase-3，CCASP-3）的表达；荧光素酶报告基因实验验证STAT-3对微RNA-21调控。建立Tscca裸鼠皮下荷瘤模型，通过免疫组织化学染色及原位末端标记（ terminal deoxynucleoitidyl transferase mediated nick end labeling ，TUNEL）法评价WP1066对细胞增殖、凋亡的作用。结果 WP1066组中STAT-3/p-STAT-3蛋白表达降低（ STAT-3：F＝15.336，P ＝0.004；p-STAT-3：F ＝52.837，P ＝0.000）；微 RNA-21表达下调（F ＝8.197，P ＝0.019）；WP1066组细胞存活率[（51±7）％]显著低于空白对照组（100％）和DMSO 组[（90±7）％]（F＝94.388，P ＝0.000）； WP1066组细胞早期凋亡率升高（F ＝217.080，P ＝0.000）；WP1066组PDCD-4、CCASP-3蛋白表达水平（0.65±0.12，0.74±0.07）比空白对照组（0.46±0.08，0.43±0.09）和DMSO 组（0.34±0.05，0.34±0.02）上调（PDCD-4：F＝8.771，P＝0.017；CCASP-3：F＝26.611，P＝0.001）；Ki-67、Bcl-2表达水平下调（Ki-67：F＝5.854，P＝0.039；Bcl-2：F＝125.502，P＝0.000）；荧光素酶报告基因实验证明 STAT-3与微 RNA-21启动子特定区域结合。体内实Objective To investigate the antitumour molecular mechanisms of WP 1066 （ STAT-3 inhibitor ） to human tongue squamous cell carcinoma in vitro and in vivo.Methods WP1066 was used to inhibit the p-STAT-3 expression in Tscca human tongue squamous cell carcinoma cell line .Real-time PCR was used to detect the microRNA-21 expression after treatment with DMSO and WP 1066.Methyl thiazolyl tatrozolium （ MTT ） assay was employed to determine cell survival.Flow cytometry （ FCM ） was used to＆amp;nbsp;measure apoptosis .The expression level of STAT-3/p-STAT-3, programmed cell death protein 4（PDCD-4）, antigen 67 （ Ki-67 ） , B cell lymphoma 2 （ Bcl-2 ） and cleaved cysteinyl aspartate specific proteinase-3 （CCASP-3） was examined by Western blotting.Luciferase reporter gene assay was conducted to verify the regulation of STAT-3 to microRNA-21.Tscca xenograft tumor model was established in BALB /c nude mice and the tumors were divided into control , DMSO and WP1066 treated groups.The tumor tissues were measured by immunohistochemistry stain and terminal-deoxynucleoitidyl transferase mediated nick end labeling （ TUNEL） assay.Results STAT-3/p-STAT-3 protein was suppressed after treatment with WP 1066 （STAT-3：F=15.336,P =0.004,p-STAT-3：F=52.837,P=0.000）.MicroRNA-21 relative expression level was down-regulated （F=8.197,P=0.019）.Cell survival rate was significantly reduced after treatment with WP1066 than control groups （ F=94.388,P=0.000）.Early apoptosis rate increased after treatment with WP1066 （ F =217.080, P =0.000 ）.PDCD-4 and cleaved cysteinyl aspartate specific proteinase-3 （CCASP-3）protein expression was increased after treatment with WP 1066（PDCD-4：F=8.771,P=0.017;CCASP-3：F=26.611,P=0.001）.Ki-67 and Bcl-2 protein was down regulated （Ki-67：F=5.854,P=0.039;Bcl-2：F=125.502,P=0.000）.Luciferase reporter gene assay proved that STAT-3 combined with specific promoter region of microRNA-21.In vivo, the tumor volume after treatment with WP 1066 was significantly

关 键 词：口腔鳞状细胞癌 舌肿瘤 细胞增殖 细胞凋亡 

分 类 号：R739.8[医药卫生—肿瘤]